Mouse submandibular gland prorenin-converting enzyme is a member of glandular kallikrein family

Mouse submandibular gland prorenin-converting enzyme (PRECE) consists of the two polypeptide chains of 17 and 10 kDa and cleaves mouse Ren-2 prorenin at a dibasic site to yield mature renin. Western blot analysis using an antiserum against this enzyme gave rise to multiple bands in mouse submandibular glands, suggesting that PRECE is a member of a protease family. Partial amino acid sequence analysis of purified PRECE and cloning and sequence analyses of its cDNA indicated that it is identical to the mGK-13 gene product, epidermal growth factor-binding protein type B, which is a member of the glandular kallikrein family and is involved in maturation of epidermal growth factor. Conditioned medium from Chinese hamster ovary cells transfected with an expression plasmid for PRECE had prorenin converting activity. These results indicate that PRECE is involved in the maturation of two bioactive polypeptides expressed in mouse submandibular glands, Ren-2 renin and epidermal growth factor.     

beta-NGF-endopeptidase: structure and activity of a kallikrein encoded by the gene mGK-22

Mouse nerve growth factor (NGF) is cleaved at a histidine-methionine bond to release an NH2-terminal octapeptide (NGF1-8). The enzyme responsible, beta-NGF-endopeptidase, is structurally and functionally similar to gamma-NGF and epidermal growth factor-binding protein (EGF-BP) and cleaves mouse low molecular weight kininogen to produce bradykinin-like activity. These data have suggested that, like gamma-NGF and EGF-BP, beta-NGF-endopeptidase is a mouse glandular kallikrein. Evidence for a physiological role for NGF1-8 encouraged studies to further characterize the structure and function of this enzyme. Purified beta-NGF-endopeptidase migrated as a single band on isoelectric focusing and reducing SDS-polyacrylamide gels. As was expected, it removed NGF1-8 from NGF. Interestingly, enzymatic activity on an artificial substrate, and on NGF, was inhibited by NGF1-8 and by bradykinin. These studies further supported the view that beta-NGF-endopeptidase acts on both NGF and kininogen. The first 30 NH2-terminal amino acids of beta-NGF-endopeptidase were sequenced. This analysis demonstrated that the enzyme is encoded by the gene designated mGK-22 (Evans et al., 1987). The sequence of this gene corresponds to that of EGF-BP type A (Anundi et al., 1982; Drinkwater et al., 1987), and so studies were performed to determine whether or not beta-NGF-endopeptidase participates in EGF complex formation. Chromatographic and kinetic data gave no evidence that beta-NGF-endopeptidase is an EGF-binding protein. Our studies suggest that contamination of high molecular weight (HMW) EGF preparations with beta-NGF-endopeptidase erroneously led to earlier designation of the product of mGK-22 as an EGF-BP.(ABSTRACT TRUNCATED AT 250 WORDS)     

The crystal structure of the mouse glandular kallikrein-13 (prorenin converting enzyme).

A crystal structure of the serine protease, mouse glandular kallikrein 13 (mGK-13) has been determined at 2.6-A resolution. This enzyme, isolated from the mouse submandibular gland, is also known as prorenin-converting enzyme and cleaves submandibular gland Ren-2 prorenin to yield active renin. The mGK-13 structure is similar to other members of the mammalian serine protease family, having five conserved disulfide bonds and an active site located in the cleft between two beta-barrel domains. The mGK-13 structure reveals for the first time an ordered kallikrein loop conformation containing a short 3(10) helix. This loop is disordered in the related porcine pancreatic kallikrein and rat submandibular tonin structures. The kallikrein loop is in close spatial proximity to the active site and is also involved in a dimeric arrangement of mGK-13. The catalytic specificity of mGK-13 for Ren-2 prorenin was studied by modeling a prorenin-derived peptide into the active site of mGK-13. This model emphasizes two electronegative substrate specificity pockets on the mGK-13 surface, which could accommodate the dibasic P2 and P1 residues at the site of prorenin cleavage by mGK-13.     

The isolation of angiotensin-converting enzyme cDNA

Angiotensin-converting enzyme (ACE) is an Zn(II)-containing dipeptidyl carboxypeptidase that converts angiotensin I to the potent vasoconstrictor, angiotensin II. Using oligonucleotide probes based on the amino acid sequence of mouse kidney ACE, cDNA encoding this protein has been isolated. One cDNA, ACE.31, encodes the N-terminal 332 amino acids of mouse ACE, a portion of the protein containing a putative 34-amino acid leader sequence and the N terminus of the mature protein. Northern analyses with cloned ACE cDNA revealed that both mouse kidney and lung express two ACE mRNAs, one of 4900 and another of 4150 bases. Southern analysis suggests that cDNA ACE.31 is the product of a single gene, and thus these data add evidence to the hypothesis that the converting enzymes produced by epithelial and endothelial cells are identical.     

Epidermal growth factor binding protein: identification of a different protein

Partial amino acid sequence analysis of epidermal growth factor binding protein (EGF-BP), an arginine esteropeptidase that specifically associates with EGF to form a high molecular weight complex in male mouse submandibular glands, has revealed a single, distinct protein that is different from three previously reported forms of EGF-BP. This protein shows substantial sequence homology with these other putative forms of EGF-BP as well as with a large family of kallikreins expressed in the mouse submandibular gland. Purified EGF-BP contains three polypeptide chains as a result of two internal cleavages at residues 87-88 and 140-141. These modifications may represent processing events that are critical for determining the binding specificity of EGF-BP, since they occur within regions surrounding the substrate binding site.     

The characterization of recombinant mouse glandular kallikreins from E. coli

A system has been developed for the expression in E. coli of 12 of the 14 expressed mouse submandibular gland kallikreins as cassettes subcloned directly from cDNA. Using the epidermal growth factor binding protein (mGK-9) and the gamma-subunit of nerve growth factor (mGK-3), as test cases, mature processed forms, obtained as functionally active proteins, as well as various precursor forms, were isolated. The expression system described allows rapid isolation of kallikrein protein from corresponding cDNA with yields of approximately 1.0 mg of purified protein from 10 g of initial cell paste. This expression system will facilitate structure/function studies of the mouse glandular kallikrein gene family and help elucidate the regions of the mature proteins responsible for the diverse catalytic behavior and growth factor interactions observed in this family of proteins.     

Identification in chickens of an evolutionarily conserved cellular ets-2 gene (c-ets-2) encoding nuclear proteins related to the products of the c-ets proto-oncogene.

In chicken cells, we previously identified a set of proteins (p58-64) structurally related to, but distinct from, the products encoded by the c-ets proto-oncogene. We report here the isolation and nucleotide sequence of a cDNA encoding nuclear products of mol. wt 58, 60, 62 and 64 kd, indistinguishable from those detected in chicken cells. The p60 and p64 species appear to represent phosphorylated versions on serine and threonine residues of p58 and p62. The homology of p58-64 to other ets-related proteins, including the v-ets encoded domain of the transforming protein of avian leukemia virus E26 and p54c-ets, the translation product of the chicken (Ck) c-ets gene, is confined to two regions of 175 and 96 amino acid residues localized respectively at the carboxy-terminal domain and close to the amino-terminal domain of these molecules. This cDNA corresponds to a gene localized in a locus distinct from that of c-ets which is transcribed as a 4.0-kb RNA species in most chicken tissues. We also identified the human (Hu) c-ets-2-encoded products as two proteins of 60 and 62 kd, highly related to chicken p58-64. This, together with the fact that the amino acid sequence of the cDNA encoding p58-64 is 95% identical to the reported partial sequence of a Hu-c-ets-2 cDNA, indicates that p58-64 are the translation products of the Ck-c-ets-2 gene.     

Mouse glandular kallikrein genes: identification and characterization of the genes encoding the epidermal growth factor binding proteins

Previously, three proteins have been separately identified as the mouse epidermal growth factor binding protein (EGF-BP). We have identified and sequenced the coding regions of three distinct genes encoding these EGF-BPs from the BALB/c strain. The genes are all members of the glandular kallikrein gene family, which encodes a highly homologous group of serine proteases. Expression of the EGF-BP genes was detected in mouse salivary gland only and was at a relatively similar level for each gene. The isolation of three distinct genes from the one mouse strain indicates that the conflicting data previously reported in the literature are not a result of allelic polymorphisms or strain differences.     

Molecular cloning and functional expression of the second mouse nm23/NDP kinase gene, nm23-M2.

A new murine cDNA of nm23/NDP kinase was isolated. A RT-PCR product was obtained from the normal mouse liver mRNA with primers designed for the human nm23-H2 gene. The product was used as a probe to screen a cDNA library from the murine melanoma cell line, B16, and two clones containing the entire open reading frame were obtained. It was predicted that the DNA sequence encoded 152 amino acids which was 98% identical to the nm23-H2 protein. The entire nm23-M1 and -M2 gene-coding regions were translated as fusion proteins with a glutathione S-transferase. These fusion proteins displayed NDP kinase activities.     

The primary structure of rat ribosomal protein S18

The amino acid sequence of the rat 40S ribosomal subunit protein S18 was deduced from the sequence of nucleotides in a recombinant cDNA. S18 has 152 amino acids and has a molecular weight of 17,707. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 10-13 copies of the S18 gene. The mRNA for the protein is about 600 nucleotides in length. Rat S18 is identical to mouse S18 (also referred to as KE3) and is related to Escherichia coli S13 and to other S13-like ribosomal proteins from Bacillus subtilis, from Bacillus stearothermophilus, and from plant mitochondria (Nicotiana tabacum and Zea mays).     

Isolation of multiple mouse cDNAs with coding homology to Saccharomyces cerevisiae CDC25: identification of a region related to Bcr, Vav, Dbl and CDC24.

In Saccharomyces cerevisiae, the product of the CDC25 gene is an essential Ras activator that appears to function by stimulating guanine nucleotide exchange on Ras. Using the ability of a mouse cDNA expression library to complement yeast cells lacking functional CDC25, Martegani et al. have identified a 1.7 kb partial cDNA from a gene, designated CDC25Mm, with homology to CDC25. We have now screened a mouse brain cDNA library to identify full-length clones of CDC25Mm. This cloning has led to the isolation of six distinct full-length cDNAs, each of which appear to be derived from the CDC25Mm gene, since their 3' 2 kb appear to be identical and to encode the same 661 C-terminal amino acids. Three cDNAs are predicted to encode protein products of 666 or 667 amino acids. The other three cDNAs encode products that are 836, 1120 and 1260 amino acids, respectively. A 241 amino acid region near the N-terminus of the two largest products was found to have homology to a domain shared by Bcr, Vav, Dbl and CDC24. Polyclonal antibodies raised to a peptide encoded by all the cDNAs have identified at least two protein products in NIH3T3 fibroblasts. Their apparent molecular weights are 75 and 95 kDa, which correspond closely to those predicted to be encoded, respectively, by the two shorter classes of cDNAs. In NIH3T3, the 95 kDa form is much more abundant than the 75 kDa form, while PC-12 pheochromocytoma cells contain relatively high levels of the 75 kDa form. We conclude that CDC25Mm is a complex gene whose protein products are regulated in a tissue-specific manner.     

Isolation and characterization of mouse MUC18 cDNA gene, and correlation of MUC18 expression in mouse melanoma cell lines with metastatic ability

The cell surface adhesion molecule human MUC18 (huMUC18 or Mel-CAM) has been postulated to play a key pathogenic role in metastatic melanoma progression. To establish an immunocompetent syngeneic mouse model that would greatly facilitate our understanding of the role of MUC18 in the metastatic behavior of melanoma, we cloned and characterized the mouse MUC18 (muMUC18) cDNA gene. The gene was amplified by RT-PCR and RACE of the poly(A)+RNA isolated from the mouse melanoma cell line B16F10/Queens. The cloned muMUC18 cDNA gene contained 28 nucleotides of 5'-UTR, 908 nucleotides of 3'-UTR, and an open reading frame (ORF) of 1947 nucleotides encoding a protein of 648 amino acids, which is two amino acids longer than huMUC18. The size of the muMUC18 mRNA is about 3 kb with a shorter 3'-UTR than the huMUC18 mRNA (about 3.3 kb). Besides, the sequence in the 3' UTR of the two mRNAs is diverse with only 31% identity. The 5'-UTR and coding sequences of the muMUC18 cDNA are 72.4 and 80.6% identical to those of huMUC18, respectively. The deduced amino acid sequence of the muMUC18 cDNA is 76.2% identical to that of huMUC18. The amino acid sequences deduced from MUC18 cDNA sequences from six other mouse melanoma cell lines are identical except one to three residues, suggesting that the muMUC18 cDNA sequence determined in this report is correct. The muMUC18 protein is predicted to be slightly more acidic than the human protein. The levels of muMUC18 mRNA and protein in nine mouse melanoma cell lines were directly proportional to their ability to establish metastatic colonies in lungs of syngeneic mice. Most biological functions of the muMUC18 may be similar to the huMUC18.     

Identification and characterization of the Egr-1 gene product, a DNA-binding zinc finger protein induced by differentiation and growth signals.

Egr-1 is an immediate-early response gene induced by diverse signals that initiate growth and differentiation. Its cDNA sequence predicts a protein with zinc fingers. We have generated an antiserum to the Egr-1 gene product and identified it as an 80-kilodalton short-lived protein in serum-stimulated mouse fibroblasts. The rat Egr-1 product has also been identified in nerve growth factor-induced PC12 cells. In addition, we show by cell fractionation and immunocytochemistry that the Egr-1 protein is located in the nucleus. We also demonstrate that it is phosphorylated. In vitro-generated Egr-1 protein binds with high affinity to the sequence CGCCCCCGC in a zinc-dependent manner.     

Human mb-1 gene: complete cDNA sequence and its expression in B cells bearing membrane Ig of various isotypes.

The transmembrane protein, IgM-alpha, a product of mb-1 gene, has been shown to be specifically associated with membrane-bound IgM on the plasma membrane of B lymphocytes. Recent studies have suggested that IgM-alpha may play a role in transducing signals from the Ag receptors during the activation of B cells. A large amount of information has been obtained in the mouse system regarding IgM-alpha and other components of the newly conceived B cell Ag receptor complex. Here we report the cloning and the nucleotide sequencing of cDNA clones of human mb-1, covering the entire length of the mRNA. At the amino acid sequence level, human and murine mb-1 share a high homology in their transmembrane and intracytoplasmic segments, suggesting an important biologic function for these regions of mb-1. A major difference, mainly in the 3' untranslated part, exists between our cDNA sequence and the published partial human mb-1 cDNA sequence. It has also been observed that human mb-1 is expressed not only by B cell lines expressing membrane-bound Ig of mu and delta isotypes but also those expressing membrane-bound Ig of alpha and gamma isotypes.